Cell penetrating peptides that inhibit irf5 nuclear localization

ABSTRACT

The invention provides inhibitors of interferon regulatory factor 5 (IRF5) nuclear localization and methods of using the inhibitors to treat autoimmune diseases such as systemic lupus erythematosus (SLE).

RELATED APPLICATION

This application claims the benefit of priority of U.S. ProvisionalApplication Ser. No. 62/215,896, filed on Sep. 9, 2015, whichapplication is incorporated by reference herein.

BACKGROUND

Systemic lupus erythematosus (SLE) is an autoimmune disease in which thebody's immune system mistakenly attacks healthy tissue. The underlyingcause of SLE is not known. SLE symptoms vary from person to person andgenerally include joint pain and swelling, with some patients developingarthritis. Unfortunately, there is no cure for SLE, and the goal oftreatment is to control symptoms. Further, side effects from certaintreatments can be severe. As such, new tools for investigating SLE andtreatments for SLE are needed.

SUMMARY OF THE INVENTION

Certain embodiments of the invention provide a polypeptide thatcomprises a cell penetrating peptide sequence and an interferonregulatory factor 5 (IRF5) nuclear localization signal (NLS) sequence.In certain embodiments, the polypeptide comprises formula (I):

X-Y-Z  (I)

wherein X is a cell penetrating peptide sequence; Y is a linking group;and Z an IRF5 NLS sequence.

As used herein, the term “cell penetrating peptide sequence” refers toany amino acid sequence that facilitates cellular intake/uptake of thepolypeptide. Cell penetrating peptide sequences typically have an aminoacid composition that either contains a high relative abundance ofpositively charged amino acids such as lysine or arginine or hassequences that contain an alternating pattern of polar/charged aminoacids and non-polar, hydrophobic amino acids. These two types ofstructures may be referred to as polycationic or amphipathic,respectively. A third class of cell penetrating peptide sequences arehydrophobic peptides, which contain only apolar residues, with low netcharge or have hydrophobic amino acid groups that are important forcellular uptake. Cell penetrating peptide sequences are known in theart, and include, but are not limited to, e.g., DRQIKIWFQNRRMKWKK (SEQID NO:5), AAVALLPAVLLALLAP (SEQ ID NO:9), GRKKRRQRRRPPQ (SEQ ID NO:10)(i.e., the HIV TAT sequence), CSIPPEVKFNKPFVYLI (SEQ ID NO:11),KKWKMRRNQFWVKVQRG (SEQ ID NO:12), KLLKLLLKLWLKLLKLLL (SEQ ID NO:13),INLKALAALAKKIL (SEQ ID NO:14), RQIKIWFQNRRMKWKKGG (SEQ ID NO:15) andGWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:16). In certain embodiments,these cell penetrating peptide sequences may be in the acid form. Incertain embodiments, these cell penetrating peptide sequences may be inthe amide form. Accordingly, in certain embodiments, the cellpenetrating peptide sequence comprises a sequence at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical toDRQIKIWFQNRRMKWKK (SEQ ID NO:5), AAVALLPAVLLALLAP (SEQ ID NO:9),GRKKRRQRRRPPQ (SEQ ID NO:10), CSIPPEVKFNKPFVYLI (SEQ ID NO: 11),KKWKMRRNQFWVKVQRG (SEQ ID NO: 12), KLLKLLLKLWLKLLKLLL (SEQ ID NO:13),INLKALAALAKKIL (SEQ ID NO:14), RQIKIWFQNRRMKWKKGG (SEQ ID NO:15) orGWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 16). In certain embodiments, thecell penetrating peptide sequence comprises DRQIKIWFQNRRMKWKK (SEQ IDNO:5), AAVALLPAVLLALLAP (SEQ ID NO:9), GRKKRRQRRRPPQ (SEQ ID NO:10),CSIPPEVKFNKPFVYLI (SEQ ID NO: 11), KKWKMRRNQFWVKVQRG (SEQ ID NO:12),KLLKLLLKLWLKLLKLLL (SEQ ID NO:13), INLKALAALAKKIL (SEQ ID NO:14),RQIKIWFQNRRMKWKKGG (SEQ ID NO:15) or GWTLNSAGYLLGKINLKALAALAKKIL (SEQ IDNO:16). In certain embodiments, the cell penetrating peptide sequence isDRQIKIWFQNRRMKWKK (SEQ ID NO:5), AAVALLPAVLLALLAP (SEQ ID NO:9),GRKKRRQRRRPPQ (SEQ ID NO:10), CSIPPEVKFNKPFVYLI (SEQ ID NO: 11),KKWKMRRNQFWVKVQRG (SEQ ID NO:12), KLLKLLLKLWLKLLKLLL (SEQ ID NO:13),INLKALAALAKKIL (SEQ ID NO:14), RQIKIWFQNRRMKWKKGG (SEQ ID NO:15) orGWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 16). In certain embodiments, thecell penetrating peptide sequence is DRQIKIWFQNRRMKWKK (SEQ ID NO:5).

As used herein, the term “interferon regulatory factor 5 (IRF5) nuclearlocalization signal (NLS) sequence” refers any sequence that can mimicIRF5 nuclear translocation signals. Two functional nuclear localizationsignals (NLS) have been identified and characterized in the IRF5 proteinthat are not conserved or homologous with NLSs in other IRFs. One NLSresides in the amino-terminus (PRRVRLK) (SEQ ID NO: 1) and the other inthe carboxyl-terminus (PREKKLI) (SEQ ID NO:2) (Barnes et al. (2002) MolCell Biol 22, 5721-5740). Accordingly, in certain embodiments, the IRF5NLS sequence comprises a sequence at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identical to PRRVRLK (SEQ ID NO:1) or PREKKLI(SEQ ID NO:2). In certain embodiments, the IRF5 NLS sequence comprisesPRRVRLK (SEQ ID NO:1) or PREKKLI (SEQ ID NO:2). In certain embodiments,the IRF5 NLS sequence is PRRVRLK (SEQ ID NO:1) or PREKKLI (SEQ ID NO:2).In certain embodiments, the IRF5 NLS sequence is PRRVRLK (SEQ ID NO:1).In certain embodiments, the IRF5 NLS sequence is PREKKLI (SEQ ID NO:2).

The nature of the linking group (Y) is not critical, and may be anygroup that can link the cell penetrating peptide sequence to the IRF5NLS sequence using known chemistry, provided that the linking group doesnot interfere with the activity of the cell penetrating peptide sequenceor the IRF5 NLS sequence (i.e., its ability to inhibit endogenous IRF5nuclear localization). For example, in certain embodiments, the linkinggroup may be a bond, such as an amide bond (i.e., a traditional peptidebond). Other linking groups include, e.g., ketomethylene (e.g.,—C(═O)—CH2- for C(═O)—NH—), aminomethylene (CH2-NH), ethylene, olefin(CH═CH), ether (CH2-O), thioether (CH2-S), tetrazole, thiazole,retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistryand Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp.267-357, “Peptide Backbone Modifications,” Marcel Dekker, N.Y.,incorporated herein by reference). In other embodiments, the linkinggroup may comprise one or more amino acids.

Certain embodiments of the present invention provide a polypeptide thatcomprises a sequence at least 90% identical to DRQIKIWFQNRRMKWKKPRRVRLK(SEQ ID NO:3), DRQIKIWFQNRRMKWKKPREKKLI (SEQ ID NO:4),DRQIKIWFQNRRMKWKKPKRRRLV (SEQ ID NO:6) or DRQIKIWFQNRRMKWKKPIKRLKE (SEQID NO:7). In certain embodiments, the polypeptide comprises a sequenceat least 90% identical to

(SEQ ID NO: 3) DRQIKIWFQNRRMKWKKPRRVRLK or (SEQ ID NO: 4)DRQIKIWFQNRRMKWICKPREKKLI.

Certain embodiments of the present invention provide a polypeptide thatis substantially identical to DRQIKIWFQNRRMKWKKPRRVRLK (SEQ ID NO:3),DRQIKIWFQNRRMKWKKPREKKLI (SEQ ID NO:4), DRQIKIWFQNRRMKWKKPKRRRLV (SEQ IDNO:6) or DRQIKIWFQNRRMKWKKPIKRLKE (SEQ ID NO:7). In certain embodiments,the polypeptide is substantially identical to DRQIKIWFQNRRMKWKKPRRVRLK(SEQ ID NO:3) or DRQIKIWFQNRRMKWKKPREKKLI (SEQ ID NO:4).

In certain embodiments, the polypeptide comprises a sequence at leastabout 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:7.

In certain embodiments, the polypeptide comprises a sequence at least95% identical to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:7.In certain embodiments, the polypeptide comprises a sequence at least95% identical to SEQ ID NO:3 or SEQ ID NO:4.

In certain embodiments, the polypeptide comprises a sequence at least99% identical to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:7.In certain embodiments, the polypeptide comprises a sequence at least99% identical to SEQ ID NO:3 or SEQ ID NO:4.

In certain embodiments, the polypeptide comprises SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:6 or SEQ ID NO:7. In certain embodiments, thepolypeptide comprises SEQ ID NO:3 or SEQ ID NO:4. In certainembodiments, the polypeptide comprises SEQ ID NO:3. In certainembodiments, the polypeptide comprises SEQ ID NO:4.

Certain embodiments of the present invention provide the polypeptide

(SEQ ID NO: 3) DRQIKIWFQNRRMKWKKPRRVRLK.

Certain embodiments of the present invention provide the polypeptide

(SEQ ID NO: 4) DRQIKIWFQNRRMKWKKPREKKLI.

Certain embodiments of the present invention provide the polypeptide

(SEQ ID NO: 6) DRQIKIWFQNRRMKWKKPKRRRLV.

Certain embodiments of the present invention provide the polypeptide

(SEQ ID NO: 7) DRQIKIWFQNRRMKWKKPIKRLKE.

In certain embodiments, the polypeptide is an inhibitor of interferonregulatory factor 5 (IRF5).

In certain embodiments, the polypeptide is an inhibitor of interferonregulatory factor 5 (IRF5) nuclear localization.

Certain embodiments of the present invention provide a nucleic acidsequence encoding a polypeptide as described herein.

Certain embodiments of the present invention provide a pharmaceuticalcomposition that comprises a polypeptide as described herein and apharmaceutically acceptable carrier.

Certain embodiments of the present invention provide a method ofinhibiting interferon regulatory factor 5 (IRF5) in an animal (e.g., amammal, such as a patient) in need thereof, comprising administering tothe patient a therapeutically effective amount of a polypeptide asdescribed herein.

Certain embodiments of the present invention provide a method ofinhibiting interferon regulatory factor 5 (IRF5) nuclear localization inan animal (e.g., a mammal, such as a patient) in need thereof,comprising administering to the patient a therapeutically effectiveamount of a polypeptide as described herein.

Certain embodiments of the present invention provide a method fortreating, e.g., an autoimmune disease, in an animal (e.g., a mammal,such as a patient) in need of such treatment, comprising administeringto the patient a therapeutically effective amount of a polypeptide asdescribed herein.

In another aspect, the present invention provides methods for treatingand/or ameliorating one or more symptoms associated an autoimmunedisease in an animal (e.g., a mammal, such as a patient) in need of suchtreatment, wherein the methods each include the step of administering tothe human a therapeutically effective amount of a polypeptide asdescribed herein.

In certain embodiments, the autoimmune disease is systemic lupuserythematosus (SLE), systemic sclerosis (scleroderma),polymyositis/dermatomyositis, Crohn's disease, rheumatoid arthritis,periodontitis, SLE-associated atherosclerosis, Sjögren's syndrome,autoimmune encephalomyelitis, sarcoidosis, Behçet's disease, myastheniagravis, lupus nephritis, inflammatory bowel disease, ankylosingspondylitis, primary biliary cirrhosis, colitis, juvenile idiopathicarthritis, pulmonary fibrosis, antiphospholipid syndrome, or psoriasis.

In certain embodiments, the autoimmune disease is systemic lupuserythematosus (SLE).

Certain embodiments of the present invention provide a method fortreating an animal (e.g., a mammal, such as a patient) having classicalHodgkin lymphoma, atherosclerosis, cardiovascular disease, neuropathicpain, or certain amenable types of leukemia and lymphoma, such as T celllarge granular lymphocyte leukemia, comprising administering to thepatient a therapeutically effective amount of a polypeptide as describedherein.

Certain embodiments of the present invention provide a polypeptide asdescribed herein for use in medical treatment or diagnosis.

Certain embodiments of the present invention provide the use of apolypeptide as described herein to prepare a medicament useful forinhibiting interferon regulatory factor 5 (IRF5) in an animal (e.g., amammal, such as a patient).

Certain embodiments of the present invention provide the use of apolypeptide as described herein to prepare a medicament useful fortreating an autoimmune disease in an animal (e.g., a mammal, such as apatient).

Certain embodiments of the present invention provide the use of apolypeptide as described herein to prepare a medicament useful fortreating classical Hodgkin lymphoma, atherosclerosis, cardiovasculardisease, neuropathic pain, or certain amenable types of leukemia andlymphoma, such as T cell large granular lymphocyte leukemia in an animal(e.g., a mammal, such as a patient).

Certain embodiments of the present invention provide a polypeptide asdescribed herein for use in therapy.

Certain embodiments of the present invention provide a polypeptide asdescribed herein for the inhibiting interferon regulatory factor 5(IRF5).

Certain embodiments of the present invention provide a polypeptide asdescribed herein for the prophylactic or therapeutic treatment of anautoimmune disease.

Certain embodiments of the present invention provide a polypeptide asdescribed herein for the prophylactic or therapeutic treatment ofclassical Hodgkin lymphoma, atherosclerosis, cardiovascular disease,neuropathic pain, or certain amenable types of leukemia and lymphoma,such as T cell large granular lymphocyte leukemia.

Certain embodiments of the present invention provide a polypeptide asdescribed herein for use in treating classical Hodgkin lymphoma,atherosclerosis, cardiovascular disease, neuropathic pain, or certainamenable types of leukemia and lymphoma, such as T cell large granularlymphocyte leukemia.

Certain embodiments of the present invention provide a pharmaceuticalcomposition for use in the treatment of an autoimmune disease,comprising a polypeptide described herein and a pharmaceuticallyacceptable carrier.

Certain embodiments of the present invention provide a pharmaceuticalcomposition for use in the treatment of treating classical Hodgkinlymphoma, atherosclerosis, cardiovascular disease, neuropathic pain, orcertain amenable types of leukemia and lymphoma, such as T cell largegranular lymphocyte leukemia, comprising a polypeptide described hereinand a pharmaceutically acceptable carrier.

Certain embodiments of the present invention provide the use of apolypeptide as described herein as a research tool, e.g., for studyinginterferon regulatory factor 5 (IRF5).

In certain embodiments of the present invention provides the use of apolypeptide as described herein for studying interferon regulatoryfactor 5 (IRF5) nuclear localization.

Certain embodiments of the present invention provide a kit comprising apolypeptide as described herein, at least one other therapeutic agent,and instructions for administering the polypeptide and the othertherapeutic agent(s) to an animal to treat an autoimmune disease,classical Hodgkin lymphoma, atherosclerosis, cardiovascular disease,neuropathic pain, or certain amenable types of leukemia and lymphoma,such as T cell large granular lymphocyte leukemia.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-E. IRF5 peptide inhibitors are cell permeable. FITC-conjugatedinhibitor titrations for cell penetrance were performed by flowcytometry analysis (FIG. 1A). Cells were co-stained with propidiumiodide to examine viability. Inhibitors have no significant effect oncell viability (FIG. 1B) or cell cycle (FIG. 1C) in Ramos B cells. FIG.1D shows representative images from imaging flow cytometry ofintracellular FITC-conjugated inhibitors in CD19+ B cells and CD14+monocytes from healthy donors. Peripheral blood mononuclear cells(PBMCs) were pre-incubated with inhibitors for 1 hour before surfacestaining and permeabilization. Nuclei were stained with DRAQ5. FIG. 1Eshows representative histogram plots of FITC-conjugated inhibitorstaining in CD14+ monocytes and CD19+ B cells using flow cytometry.

FIGS. 2A-E. Peptides (non-FITC-conjugated) inhibit R848-induced IRF5nuclear translocation. Healthy donor PBMCs were pre-incubated withpeptide inhibitors for 1 hour and then stimulated for 2 hours with theToll-like receptor agonist R848. IRF5 cellular localization wasdetermined in CD14+ monocytes (FIG. 2A) and CD19+ B cells (FIG. 2B) bystaining with intracellular IRF5 and DRAQ5. Similarity scores betweenIRF5 and DRAQ5 were determined on the Amnis Imagestream using IDEASsoftware. FIG. 2C shows representative images from FIG. 2B. FIG. 2Dshows a representative Western blot of nuclear-localized IRF5 usingnuclear extracts from purified CD14+ healthy donor monocytes. Lamin B1serves as a nuclear protein loading control. Cells were pre-incubatedwith the indicated inhibitors for 1 hour and then stimulated with LPSfor 2 hours. FIG. 2E shows results from densitometry analysis of nuclearIRF5 in FIG. 2D. IRF5 band intensity was normalized by Lamin B1, andrelative fold change of IRF5 in nuclear portion was calculated over PBStreated sample. In FIGS. 2A-B and E, the bar on the left is mock (NT)and the bar on the right is R848.

FIGS. 3A-B. Peptide inhibitors are specific for IRF5. Human healthydonor PBMCs were pre-incubated with the indicated inhibitors for 1 hourand cells stimulated with R848 for 30 min (FIG. 3A) or 2 hours (FIG.3B). NFκB nuclear translocation was determined in CD14+ monocytes (FIG.3A) and IRF7 nuclear translocation in BDCA2+CD123+ plasmacytoiddendritic cells by imaging flow cytometry.

FIGS. 4A-C. IRF5 peptide inhibitors inhibit R848-induced proinflammatorycytokine expression in PBMCs. The expression of IL6 (FIG. 4A), IL10(FIG. 4B), and IFNA (FIG. 4C) was determined by quantitative real-timePCR after 6 hours stimulation of PBMCs with R848.

FIG. 5. IRF5 peptide inhibitors inhibit SLE serum-induced IRF5 nucleartranslocation. Healthy donor PBMCs were pre-incubated with peptideinhibitors for 1 hour and then stimulated with SLE serum for 2 hours.IRF5 nuclear localization was determined in CD14+ monocytes by imagingflow cytometry.

FIGS. 6A-B. Inhibition of nuclear translocation of murine IRF5 by IRF5peptide inhibitor (N′terminal) in response to LPS stimulation. (FIG. 6A)RAW264.6 cells were treated with PBS or 5 μg/mL LPS for 2 hours.Scramble (Scr) or N′terminal were pre-incubated at the indicatedconcentrations for 1 hour before stimulation with LPS. Nuclear extractswere subjected to Western blotting with anti-IRF5 and anti-Lamin B1antibodies. (FIG. 6B) IRF5 band intensity was normalized to Lamin B1,and relative fold change of IRF5 in nuclear portion was calculated overPBS treated sample.

FIGS. 7A-B. IRF5 N′terminal inhibitor protects NZB/W F1 from in vivopathogenic autoantibody production. 8 week-old NZB/W mice weremock-injected or IP-injected over 2 weeks with 100 μg IRF5 peptideinhibitor. Autoantibodies were analyzed by testing sera (1:100 dilution)on HEp-2 cells from age-matched mock or treated NZB/W mice (FIG. 7A).Three out of six mice in control group (Ctrl) were positive and zero outof six in 100 μg-treated group (100 μg) were positive at week 27.Representative pictures from control and treated group were taken at thesame exposure time (FIG. 7B).

FIGS. 8A-B. IRF5 peptide inhibitor reduces proteinuria in NZB/W mice.Proteinuria was measured by Bradford protein assay. NZB/W F1 mice wereIP-injected with 100 μg/day N′terminal (100 μg, ●) or 100 μl PBS (Ctrl,□) at 8-weeks of age (early onset) (FIG. 8A) or 27-weeks of age (lateonset) (FIG. 8B), on day 0, 1, 4, 7 and 14. * p<0.05 vs Ctrl; n=6/group.

FIG. 9. Delta change of body weight at week 34. NZB/W F1 mice wereIP-injected with 100 μg/day N′terminal (100 μg) or 100 μl PBS (Ctrl) at8 weeks-old, on day 0, 1, 4, 7 and 14 (n=6/group). The delta change ofbody weight was calculated at week 34 in comparison to baseline (week8).

FIG. 10. Comparisons of percent survival between n=6 NZB/W F1 micetreated with N′terminal peptide and n=6 mock-treated mice. NZB/W F1 micewere IP injected with 100 μg/day N′terminal (100 μg) or 100 μl PBS(Ctrl) at 27-week age, on day 0, 1, 4, 7 and 14. Percentage of survivalwas sensed until week 40.

FIG. 11. Specific binding of the 5′-terminal inhibitor to humanrecombinant full-length IRF5. 20 μg/mL recombinant IRF5 was immobilizedon the Biacore Sensor Chip CM5 at a flow rate of 10 μL/min using manualinjection (450RU). Running buffer was filtered 1×PBS with 0.05% P20 runat a flow rate of 30 μl/min. Contact time—60 sec; Dissociation time—120sec. Samples were run on a Biacore-T200.

DETAILED DESCRIPTION

Systemic Lupus Erythematosus (SLE)

Systemic lupus erythematosus (SLE) is an autoimmune disease in which thebody's immune system mistakenly attacks healthy tissue. SLE is morecommon in women than men. SLE may occur at any age but appears mostoften in people between the ages of 10 and 50. African Americans andAsians are affected more often than people from other races. SLE canaffect the skin, joints, kidneys, brain, and other organs. Theunderlying cause of SLE is not fully understood. Symptoms vary fromperson to person, and may come and go. Almost everyone with SLE hasjoint pain and swelling, and some develop arthritis. The joints of thefingers, hands, wrists, and knees are often affected. Other commonsymptoms include: chest pain when taking a deep breath, fatigue, feverwith no other cause, general discomfort, uneasiness, or ill feeling(malaise), hair loss, mouth sores, sensitivity to sunlight, skin rash (a“butterfly” rash occurs in about half people with SLE. The rash is mostoften seen over the cheeks and bridge of the nose, but can bewidespread. It typically gets worse in sunlight.), and swollen lymphnodes. Other symptoms depend on which part of the body is affected:brain and nervous system; headaches, numbness, tingling, seizures,vision problems, personality changes; digestive tract: abdominal pain,nausea, and vomiting; heart: abnormal heart rhythms (arrhythmias); lung:coughing up blood and difficulty breathing; skin: patchy skin color,fingers that change color when cold (Raynaud's phenomenon); kidney:swelling in the legs, weight gain.

Unfortunately, there is no cure for SLE. The goal of treatment is tocontrol symptoms. Severe symptoms that involve the heart, lungs,kidneys, and other organs often need treatment from specialists. Mildforms of the disease may be treated with: NSAIDs for joint symptoms andpleurisy; corticosteroid creams for skin rashes; a drug also used totreat malaria (hydroxychloroquine); and low-dose corticosteroids forskin and arthritis symptoms. Treatments for more severe SLE may include:high-dose corticosteroids and cytotoxic drugs (drugs that block cellgrowth or drugs which dampen or suppress the immune system). Sideeffects from these drugs can be severe.

Interferon regulatory factor 5 (IRF5) Interferon regulatory factor 5(IRF5) is a transcription factor that regulates key signaling pathwaysthat result in proinflammatory cytokine expression, including but notlimited to type I interferons, interleukin (IL)-12, IL-6 and tumornecrosis factor (TNF)-α. Numerous genome wide association studies (GWAS)have reported that IRF5 polymorphisms are associated with an increasedrisk of the autoimmune disease SLE (Xu et al., (2013) Expert Rev Mol Med15:e6. doi: 10.1017/erm.2013.7). IRF5 has become a putative target forthe regulation of autoimmune pathology. Pre-clinical data providescompelling rationale that blocking IRF5 function may be beneficial toSLE patients as IRF5 expression is upregulated and the proteinoveractivated in primary immune cells from SLE patients (Feng et al.(2010) Arthritis Rheum 62, 562-573; Stone et al. (2012) Arthritis Rheum64:788-798; Stone et al. (2013) PLoS ONE 8:e54487; Niewold et al. (2008)Arthritis Rheum 58, 2481-2487; Hedl M, Abraham C. (2012) J Immunol. 188,5348-5356). The only current tools available for pre-clinical targetevaluation are the use of siRNA targeting IRF5 or Irf5 transgenicknockout mice. Importantly, published studies from Irf5 knockout miceconfirm that loss of Irf5 expression, and therefore function, protectsmice from SLE disease onset, supporting the hypothesis that inhibitingIRF5 function will be beneficial to SLE patients (Feng et al. (2012) EurJ Immunol 42(6):1477-87; Yang et al. (2012) J Immunol 189:3741-3750).

Human IRF5 is constitutively expressed in cells of the immune system,particularly plasmacytoid dendritic cells, monocytes, andmonocyte-derived dendritic cells, as well as activated B cells. IRF5exists as multiple alternatively spliced variants resulting in theexpression of a variety of isoforms each with distinct regulation,cellular localization and function. IRF5 is primarily a cytoplasmicprotein in unstimulated cells that becomes post-translationally modifiedafter stimulation with virus, Toll-like receptor ligands, DNA damage orTNF-related apoptosis inducing ligand (TRAIL) resulting in nucleartranslocation and the induction of IRF5 target genes (Barnes et al.(2002) Mol Cell Biol 22, 5721-5740). Upon post-translationalmodification, IRF5 forms homodimers which have also been considered toreflect “activation”; however, homodimer formation does not ensure IRF5nuclear translocation (Cheng et al. (2006) J Immunol 176, 7462-7470;Foreman et al. (2012) PLoS ONE DOI: 10.1371/journal.pone.0033098). IRF5is only truly “activated” and functional once it translocates from thecytoplasm to the nucleus (Barnes et al. (2002) Mol Cell Biol 22,5721-5740). IRF5 has also been shown to regulate the expression ofcytokines/chemokines with lymphocyte-chemotactic activities, e.g.,RANTES, MIP1α/β, MCP-1, I-309, IL8, IP10, and CXCL13, and to mediatecellular apoptosis.

Interferon regulatory factor 5 (IRF5) polymorphisms as well as IRF5activity are considered viable markers of systemic lupus erythematosus(SLE) disease activity and severity. IRF5 is constitutively activated inSLE monocytes (Stone et al. (2012) Arthritis Rheum 64:788-798) and inSLE B cells. IRF5 is proposed to be a viable target in other autoimmunediseases. The inhibitors described herein are useful to study IRF5function in primary human immune cells and to determine the potentialeffects of inhibiting IRF5 in patients. As described herein, IRF5polymorphisms contribute to risk of numerous autoimmune diseases,including but not limited to rheumatoid arthritis, inflammatory bowelsyndrome and multiple sclerosis. As such, tools that specifically targetIRF5 activation and therefore function, will be valuable across numerousfields and diseases, for example, for studying and/or treatingautoimmune diseases such as systemic lupus erythematosus (SLE), systemicsclerosis (scleroderma), polymyositis/dermatomyositis, Crohn's disease,rheumatoid arthritis, periodontitis, SLE-associated atherosclerosis,Sjögren's syndrome, autoimmune encephalomyelitis, sarcoidosis, Behçet'sdisease, myasthenia gravis, lupus nephritis, inflammatory bowel disease,ankylosing spondylitis, primary biliary cirrhosis, colitis, juvenileidiopathic arthritis, pulmonary fibrosis, antiphospholipid syndrome andpsoriasis. Other non-autoimmune diseases that may be studied and/ortreated include classical Hodgkin lymphoma, atherosclerosis,cardiovascular disease, neuropathic pain, and some types of leukemia andlymphoma, such as T cell large granular lymphocyte leukemia.

As described in the Examples, the inhibitors have been evaluated in avariety of different cellular assays, as well as in an animal model ofSLE (see, e.g., Heyler and Howie, Nature (1963) 4863:197; Theofilopoulosand Dixon, Adv Immunol (1985) 37:269-390). The inhibitors are selectivefor IRF5 and not other IRF family members. The stability of theinhibitors has been examined over time in cell culture and are found tobe stable over 72 hours. FITC-conjugated inhibitors are detected at 72hours post-incubation in PBMCs.

As described herein, the inhibitors comprise a cell penetration sequenceand an IRF5 NLS sequence. Accordingly, these inhibitors arenon-naturally occurring peptides that are not products of nature.Additionally, the inhibitors described herein comprise markedlydifferent characteristics (e.g., structural, functional and/or otherproperties) as compared to naturally occurring peptides that comprise anIRF5 NLS sequence or a cell penetration sequence.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form, made of monomers (nucleotides) containing a sugar,phosphate and a base that is either a purine or pyrimidine. Unlessspecifically limited, the term encompasses nucleic acids containingknown analogs of natural nucleotides that have similar bindingproperties as the reference nucleic acid and are metabolized in a mannersimilar to naturally occurring nucleotides. Unless otherwise indicated,a particular nucleic acid sequence also encompasses conservativelymodified variants thereof (e.g., degenerate codon substitutions) andcomplementary sequences, as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues.

The term “nucleotide sequence” refers to a polymer of DNA or RNA thatcan be single-stranded or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases capable ofincorporation into DNA or RNA polymers. The terms “nucleic acid,”“nucleic acid molecule,” or “polynucleotide” are used interchangeably.

By “portion” or “fragment,” as it relates to a nucleic acid molecule,sequence or segment of the invention, when it is linked to othersequences for expression, is meant a sequence having at least 80nucleotides, more preferably at least 150 nucleotides, and still morepreferably at least 400 nucleotides. If not employed for expressing, a“portion” or “fragment” means at least 9, preferably 12, more preferably15, even more preferably at least 20, consecutive nucleotides, e.g.,probes and primers (oligonucleotides), corresponding to the nucleotidesequence of the nucleic acid molecules of the invention.

Certain embodiments of the invention encompass isolated or substantiallypurified nucleic acid compositions. In the context of the presentinvention, an “isolated” or “purified” DNA molecule or RNA molecule is aDNA molecule or RNA molecule that exists apart from its nativeenvironment and is therefore not a product of nature. An isolated DNAmolecule or RNA molecule may exist in a purified form or may exist in anon-native environment such as, for example, a transgenic host cell. Forexample, an “isolated” or “purified” nucleic acid molecule issubstantially free of other cellular material or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized. In oneembodiment, an “isolated” nucleic acid is free of sequences thatnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived.

The term “amino acid” includes the residues of the natural amino acids(e.g., Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Hyl, Hyp, Ile, Leu,Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D or L form, as wellas unnatural amino acids (e.g., phosphoserine, phosphothreonine,phosphotyrosine, hydroxyproline, gamma-carboxyglutamate; hippuric acid,octahydroindole-2-carboxylic acid, statine,1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine,ornithine, citruline, α-methyl-alanine, para-benzoylphenylalanine,phenylglycine, propargylglycine, sarcosine, and tert-butylglycine). Theterm also comprises natural and unnatural amino acids bearing aconventional amino protecting group (e.g., acetyl or benzyloxycarbonyl),as well as natural and unnatural amino acids protected at the carboxyterminus (e.g., as a (C₁-C₆)alkyl, phenyl or benzyl ester or amide; oras an α-methylbenzyl amide). Other suitable amino and carboxy protectinggroups are known to those skilled in the art (See for example, T. W.Greene, Protecting Groups In Organic Synthesis; Wiley: New York, 1981,and references cited therein).

“Amino acid” or “amino acid sequence” include an oligopeptide, peptide,polypeptide, or protein sequence, or to a fragment, portion, or subunitof any of these, and to naturally occurring or synthetic molecules. Theterms “polypeptide” and “protein” include amino acids joined to eachother by peptide bonds or modified peptide bonds, i.e., peptideisosteres, and may contain modified amino acids other than the 20gene-encoded amino acids. The term “polypeptide” also includes peptidesand polypeptide fragments, motifs and the like. Capitalized,single-letter abbreviations of the amino acids refer to the naturalL-isomer. Lower case, single-letter abbreviations of the amino acidsdenotes the D-isomer.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably to refer to polymers of amino acids of any length. Incertain embodiments, peptides and polypeptides may be entirely composedof natural peptide amino acids, be entirely composed of synthetic,non-natural analogues of amino acids, or, may be a chimeric molecule ofpartly natural peptide amino acids and partly non-natural analogs ofamino acids. In one aspect, a polypeptide is used in a composition, cellsystem or process of the invention. In addition, polypeptide can referto compounds comprised of polymers of amino acids covalently attached toanother functional group (e.g., a label, solubilizing group, a targetinggroup, PEG, non-amino acid group, or other therapeutic agent). Incertain embodiments, a polypeptide of the invention may be operablylinked to a label (e.g., through a direct bond or through a linkinggroup), such as a fluorescent label or a radiolabel; the labeledpeptides may be used for diagnostic imaging, research or therapeuticpurposes. Unless stated otherwise, peptide sequences are shown with theN′terminus on the left and the C′terminus on the right.

The invention encompasses isolated or substantially purified proteincompositions. In the context of the present invention, an “isolated” or“purified” polypeptide is a polypeptide that exists apart from itsnative environment and is therefore not a product of nature. The terms“polypeptide” and “protein” are used interchangeably herein. An isolatedprotein molecule may exist in a purified form or may exist in anon-native environment such as, for example, a transgenic host cell orbacteriophage. For example, an “isolated” or “purified” protein, orbiologically active portion thereof, is substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. A protein that is substantiallyfree of cellular material includes preparations of protein orpolypeptide having less than about 30%, 20%, 10%, 5%, (by dry weight) ofcontaminating protein. When the protein of the invention, orbiologically active portion thereof, is recombinantly produced,preferably culture medium represents less than about 30%, 20%, 10%, or5% (by dry weight) of chemical precursors or non-protein-of-interestchemicals. Fragments and variants of the disclosed proteins orpartial-length proteins encoded thereby are also encompassed by thepresent invention. By “fragment” or “portion” is meant a full length orless than full length of the amino acid sequence of a protein.

The genes and nucleotide sequences of the invention include both thenaturally occurring sequences as well as mutant forms. Likewise, thepolypeptides of the invention encompass naturally occurring proteins aswell as variations and modified forms thereof. Such variants willcontinue to possess the desired activity. The deletions, insertions, andsubstitutions of the polypeptide sequence encompassed herein are notexpected to produce radical changes in the characteristics of thepolypeptide. However, when it is difficult to predict the exact effectof the substitution, deletion, or insertion in advance of doing so, oneskilled in the art will appreciate that the effect will be evaluated byroutine screening assays.

Individual substitutions deletions or additions that alter, add ordelete a single amino acid or a small percentage of amino acids(typically less than 5%, more typically less than 1%) in an encodedsequence are “conservatively modified variations,” where the alterationsresult in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. The following five groupseach contain amino acids that are conservative substitutions for oneanother: Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L),Isoleucine (I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan(W); Sulfur-containing: Methionine (M), Cysteine (C); Basic: Arginine(R), Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid(E), Asparagine (N), Glutamine (Q). In addition, individualsubstitutions, deletions or additions which alter, add or delete asingle amino acid or a small percentage of amino acids in an encodedsequence are also “conservatively modified variations.”

Polypeptide compositions of the invention can contain any combination ofnon-natural structural components. Accordingly, a polypeptide of theinvention may comprise a chemical modification. For example, thepolypeptide may comprise one or more synthetic non-natural peptide aminoacids. Additionally, the linking group joining individual peptideresidues or the peptide scaffold may be chemically modified as describedbelow. Individual peptide residues can be joined by peptide bonds, otherchemical bonds or coupling means, such as, e.g., glutaraldehyde,N-hydroxysuccinimide esters, bifunctional maleimides,N,N′-dicyclohexylcarbodiimide (DCC) or N,N′-diisopropylcarbodiimide(DIC). Linking groups that can be an alternative to the traditionalamide bond (“peptide bond”) linkages include, e.g., ketomethylene (e.g.,—C(═O)—CH2- for —C(═O)—NH—), aminomethylene (CH2-NH), ethylene, olefin(CH═CH), ether (CH2-O), thioether (CH2-S), tetrazole, thiazole,retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistryand Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp.267-357, “Peptide Backbone Modifications,” Marcel Dekker, N.Y.,incorporated herein by reference). Non-natural scaffolds may also beused to stabilize a polypeptide described herein. For example,hydrocarbon bridges can be used to crosslink side chains(hydrocarbon-stapled or hydrogen bond surrogate alpha-helices);terphenyls may be added, beta amino acids may be included to generate abeta-peptide and/or a peptoid, mini protein may be generated.Accordingly, in certain embodiments of the invention, a polypeptidedescribed herein may be chemically modified (i.e., comprise anon-natural structural component). In certain embodiments, the chemicalmodification stabilizes the polypeptide.

Polypeptides used to practice methods of the invention can be modifiedby either natural processes, such as post-translational processing(e.g., phosphorylation, acylation, etc), or by chemical modificationtechniques, which result in a modified polypeptide. Modificationsdiscussed herein can occur anywhere in the polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxylterminus. It will be appreciated that the same type of modification maybe present in the same or varying degrees at several sites in a givenpolypeptide. Also a given polypeptide may have many types ofmodifications. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of a phosphatidylinositol, cross-linkingcyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,PEGylation, proteolytic processing, phosphorylation, prenylation,selenoylation, sulfation, and transfer-RNA mediated addition of aminoacids to protein such as arginylation. See, e.g., Creighton, T. E.,Proteins-Structure and Molecular Properties 2nd Ed., W. H. Freeman andCompany, New York (1993); Posttranslational Covalent Modification ofProteins, B. C. Johnson, Ed., Academic Press, New York, pp. 1-12 (1983),incorporated herein by reference.

The following terms are used to describe the sequence relationshipsbetween two or more sequences: (a) “reference sequence,” (b) “comparisonwindow,” (c) “sequence identity,” (d) “percentage of sequence identity,”and (e) “substantial identity.”

-   -   (a) As used herein, “reference sequence” is a defined sequence        used as a basis for sequence comparison. A reference sequence        may be a subset or the entirety of a specified sequence; for        example, as a segment of a full-length cDNA, gene sequence or        peptide sequence, or the complete cDNA, gene sequence or peptide        sequence.    -   (b) As used herein, “comparison window” makes reference to a        contiguous and specified segment of a sequence, wherein the        sequence in the comparison window may comprise additions or        deletions (i.e., gaps) compared to the reference sequence (which        does not comprise additions or deletions) for optimal alignment        of the two sequences. Generally, the comparison window is at        least 20 contiguous nucleotides in length, and optionally can be        30, 40, 50, 100, or longer. Those of skill in the art understand        that to avoid a high similarity to a reference sequence due to        inclusion of gaps in the sequence a gap penalty is typically        introduced and is subtracted from the number of matches.

Methods of alignment of sequences for comparison are well-known in theart. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (Myers and Miller, CABIOS, 4, 11 (1988)); the localhomology algorithm of Smith et al. (Smith et al., Adv. Appl. Math., 2,482 (1981)); the homology alignment algorithm of Needleman and Wunsch(Needleman and Wunsch, J M B, 48, 443 (1970)); thesearch-for-similarity-method of Pearson and Lipman (Pearson and Lipman,Proc. Natl. Acad. Sci. USA, 85, 2444 (1988)); the algorithm of Karlinand Altschul (Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 87, 2264(1990)), modified as in Karlin and Altschul (Karlin and Altschul, Proc.Natl. Acad. Sci. USA 90, 5873 (1993)).

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL; the ALIGNprogram and GAP, BESTFIT, BLAST, FASTA, and TFASTA. Alignments usingthese programs can be performed using the default parameters. TheCLUSTAL program is well described by Higgins et al. (Higgins et al.,CABIOS, 5, 151 (1989)); Corpet et al. (Corpet et al., Nucl. Acids Res.,16, 10881 (1988)); Huang et al. (Huang et al., CABIOS, 8, 155 (1992));and Pearson et al. (Pearson et al., Meth. Mol. Biol., 24, 307 (1994)).

Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. In addition tocalculating percent sequence identity, the BLAST algorithm also performsa statistical analysis of the similarity between two sequences.

One measure of similarity provided by the BLAST algorithm is thesmallest sum probability (P(N)), which provides an indication of theprobability by which a match between two nucleotide or amino acidsequences would occur by chance. For example, a test nucleic acidsequence is considered similar to a reference sequence if the smallestsum probability in a comparison of the test nucleic acid sequence to thereference nucleic acid sequence is less than about 0.1, less than about0.01, or even less than about 0.001.

To obtain gapped alignments for comparison purposes, Gapped BLAST can beutilized. Alternatively, PSI-BLAST can be used to perform an iteratedsearch that detects distant relationships between molecules. Whenutilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of therespective programs (e.g., BLASTN for nucleotide sequences, BLASTX forproteins) can be used. Alignment may also be performed manually byinspection.

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences makes reference to a specifiedpercentage of residues in the two sequences that are the same whenaligned for maximum correspondence over a specified comparison window,as measured by sequence comparison algorithms or by visual inspection.When percentage of sequence identity is used in reference to proteins itis recognized that residue positions which are not identical oftendiffer by conservative amino acid substitutions, where amino acidresidues are substituted for other amino acid residues with similarchemical properties (e.g., charge or hydrophobicity) and therefore donot change the functional properties of the molecule. When sequencesdiffer in conservative substitutions, the percent sequence identity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Sequences that differ by such conservative substitutionsare said to have “sequence similarity” or “similarity.” Means for makingthis adjustment are well known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison, andmultiplying the result by 100 to yield the percentage of sequenceidentity.

(e) (i) The term “substantial identity” of sequences means that amolecule comprises a sequence that has at least 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, or 94%, or even at least 95%, 96%, 97%, 98%, or99% sequence identity, compared to a reference sequence using one of thealignment programs described using standard parameters. One of skill inthe art will recognize that these values can be appropriately adjustedto determine corresponding identity of proteins encoded by twonucleotide sequences by taking into account codon degeneracy, amino acidsimilarity, reading frame positioning, and the like.

The term “substantial identity” in the context of a peptide indicatesthat a peptide comprises a sequence with at least 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, or 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, or 89%, at least 90%, 91%, 92%, 93%, or 94%, or 95%, 96%, 97%, 98%or 99%, sequence identity to the reference sequence over a specifiedcomparison window. An indication that two peptide sequences aresubstantially identical is that one peptide is immunologically reactivewith antibodies raised against the second peptide. Thus, a peptide issubstantially identical to a second peptide, for example, where the twopeptides differ only by a conservative substitution.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength and pH. However, stringent conditions encompasstemperatures in the range of about 1° C. to about 20° C., depending uponthe desired degree of stringency as otherwise qualified herein. Nucleicacids that do not hybridize to each other under stringent conditions arestill substantially identical if the polypeptides they encode aresubstantially identical. This may occur, e.g., when a copy of a nucleicacid is created using the maximum codon degeneracy permitted by thegenetic code. One indication that two nucleic acid sequences aresubstantially identical is when the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid.

The phrase “hybridizing specifically to” refers to the binding,duplexing, or hybridizing of a molecule only to a particular nucleotidesequence under stringent conditions when that sequence is present in acomplex mixture (e.g., total cellular) DNA or RNA. “Bind(s)substantially” refers to complementary hybridization between a probenucleic acid and a target nucleic acid and embraces minor mismatchesthat can be accommodated by reducing the stringency of the hybridizationmedia to achieve the desired detection of the target nucleic acidsequence.

Certain embodiments of the invention provide an expression cassettecomprising a nucleic acid molecule described herein. In certainembodiments, the expression cassette described herein further comprisesa promoter, such as a regulatable promoter or a constitutive promoter.Examples of suitable promoters include a CMV, RSV, pol II or pol IIIpromoter. The expression cassette may further contain a polyadenylationsignal (such as a synthetic minimal polyadenylation signal) and/or amarker gene. Certain embodiments of the invention provide a vectorcomprising an expression cassette described herein.

A “vector” is defined to include, inter alia, any viral vector, as wellas any plasmid, cosmid, phage or binary vector in double or singlestranded linear or circular form that may or may not be selftransmissible or mobilizable, and that can transform prokaryotic oreukaryotic host either by integration into the cellular genome or existextrachromosomally (e.g., autonomous replicating plasmid with an originof replication).

“Expression cassette” as used herein means a nucleic acid sequencecapable of directing expression of a particular nucleotide sequence inan appropriate host cell, which may include a promoter operably linkedto the nucleotide sequence of interest that may be operably linked totermination signals. The coding region usually codes for a functionalpeptide of interest, for example a polypeptide described herein. Theexpression cassette including the nucleotide sequence of interest may bechimeric. The expression cassette may also be one that is naturallyoccurring but has been obtained in a recombinant form useful forheterologous expression. The expression of the nucleotide sequence inthe expression cassette may be under the control of a constitutivepromoter or of a regulatable promoter that initiates transcription onlywhen the host cell is exposed to some particular stimulus. In the caseof a multicellular organism, the promoter can also be specific to aparticular tissue or organ or stage of development.

Such expression cassettes can include a transcriptional initiationregion linked to a nucleotide sequence of interest. Such an expressioncassette is provided with a plurality of restriction sites for insertionof the gene of interest to be under the transcriptional regulation ofthe regulatory regions. The expression cassette may additionally containselectable marker genes.

“Regulatory sequences” are nucleotide sequences located upstream (5′non-coding sequences), within, or downstream (3′ non-coding sequences)of a coding sequence, and which influence the transcription, RNAprocessing or stability, or translation of the associated codingsequence. Regulatory sequences include enhancers, promoters, translationleader sequences, introns, and polyadenylation signal sequences. Theyinclude natural and synthetic sequences as well as sequences that may bea combination of synthetic and natural sequences. As is noted above, theterm “suitable regulatory sequences” is not limited to promoters.However, some suitable regulatory sequences useful in the presentinvention will include, but are not limited to constitutive promoters,tissue-specific promoters, development-specific promoters, regulatablepromoters and viral promoters.

“5′ non-coding sequence” refers to a nucleotide sequence located 5′(upstream) to the coding sequence. It is present in the fully processedmRNA upstream of the initiation codon and may affect processing of theprimary transcript to mRNA, mRNA stability or translation efficiency(Turner et al., 1995).

“3′ non-coding sequence” refers to nucleotide sequences located 3′(downstream) to a coding sequence and may include polyadenylation signalsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor.

“Promoter” refers to a nucleotide sequence, usually upstream (5′) to itscoding sequence, which directs and/or controls the expression of thecoding sequence by providing the recognition for RNA polymerase andother factors required for proper transcription. “Promoter” includes aminimal promoter that is a short DNA sequence comprised of a TATA-boxand other sequences that serve to specify the site of transcriptioninitiation, to which regulatory elements are added for control ofexpression. “Promoter” also refers to a nucleotide sequence thatincludes a minimal promoter plus regulatory elements that is capable ofcontrolling the expression of a coding sequence or functional RNA. Thistype of promoter sequence consists of proximal and more distal upstreamelements, the latter elements often referred to as enhancers.Accordingly, an “enhancer” is a DNA sequence that can stimulate promoteractivity and may be an innate element of the promoter or a heterologouselement inserted to enhance the level or tissue specificity of apromoter. It is capable of operating in both orientations (normal orflipped), and is capable of functioning even when moved either upstreamor downstream from the promoter. Both enhancers and other upstreampromoter elements bind sequence-specific DNA-binding proteins thatmediate their effects. Promoters may be derived in their entirety from anative gene, or be composed of different elements derived from differentpromoters found in nature, or even be comprised of synthetic DNAsegments. A promoter may also contain DNA sequences that are involved inthe binding of protein factors that control the effectiveness oftranscription initiation in response to physiological or developmentalconditions. Examples of promoters that may be used in the presentinvention include the mouse U6 RNA promoters, synthetic human H1RNApromoters, SV40, CMV, RSV, RNA polymerase II and RNA polymerase IIIpromoters.

In one embodiment, the phrase “selectively binds” means that apolypeptide made or used in the present invention preferentially bindsto one type of interferon regulatory factor (IRF) over another type whenin the presence of a mixture of two or more forms of IRF (e.g., apolypeptide selectively binds to IRF5 over another IRF form).

“Operably-linked” refers to the association two chemical moieties sothat the function of one is affected by the other, e.g., an arrangementof elements wherein the components so described are configured so as toperform their usual function.

“Systemic delivery,” as used herein, refers to delivery that leads to abroad biodistribution of an active agent (e.g., the inhibitors describedherein) within an organism. Some techniques of administration can leadto the systemic delivery of certain agents, but not others. Systemicdelivery means that a useful, preferably therapeutic, amount of an agentis exposed to most parts of the body. To obtain broad biodistributiongenerally requires a blood lifetime such that the agent is not rapidlydegraded or cleared (such as by first pass organs (liver, lung, etc.) orby rapid, nonspecific cell binding) before reaching a disease sitedistal to the site of administration. Systemic delivery of agents can beby any means known in the art including, for example, intravenous,subcutaneous, and intraperitoneal. In a preferred embodiment, systemicdelivery is by intravenous delivery.

“Local delivery,” as used herein, refers to delivery of an active agentsuch to a target site within an organism. For example, an agent can belocally delivered by direct injection into a disease site, other targetsite, or a target organ such as the liver, heart, pancreas, kidney, andthe like.

The terms “treat” and “treatment” refer to both therapeutic treatmentand prophylactic or preventative measures, wherein the object is toprevent or decrease an undesired physiological change or disorder, suchas the development of an autoimmune disease or other disease/disorderdiscussed herein. For purposes of this invention, beneficial or desiredclinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. “Treatment” canalso mean prolonging survival as compared to expected survival if notreceiving treatment. Those in need of treatment include those alreadywith the condition or disorder as well as those prone to have thecondition or disorder or those in which the condition or disorder is tobe prevented.

Administration

A polypeptide of the invention can be formulated as a pharmaceuticalcomposition and administered to a mammalian host, such as a humanpatient in a variety of forms adapted to the chosen route ofadministration, i.e., orally or parenterally, by intravenous,intramuscular, topical or subcutaneous routes.

Thus, the present polypeptides may be systemically administered, e.g.,orally (e.g., added to drinking water), in combination with apharmaceutically acceptable vehicle such as an inert diluent or anassimilable edible carrier. They may be enclosed in hard or soft shellgelatin capsules, may be compressed into tablets, or may be incorporateddirectly with the food of the patient's diet. For oral therapeuticadministration, the polypeptide may be combined with one or moreexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations should contain at least 0.1% of thepolypeptide. The percentage of the compositions and preparations may, ofcourse, be varied and may conveniently be between about 2 to about 60%of the weight of a given unit dosage form. The amount of polypeptide insuch therapeutically useful compositions is such that an effectivedosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the polypeptide, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the polypeptide may beincorporated into sustained-release preparations and devices.

The polypeptide may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the polypeptideor its salts can be prepared in water, optionally mixed with a nontoxicsurfactant. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, triacetin, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations contain apreservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient, which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form should be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating thepolypeptide in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filter sterilization. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and the freeze-drying techniques, whichyield a powder of the active ingredient plus any additional desiredingredient present in the previously sterile-filtered solutions.

For topical administration, the present polypeptides may be applied inpure form, i.e., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the present polypeptides can be dissolved or dispersedat effective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions, which can be used todeliver the polypeptides to the skin, are known to the art; for example,see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No.4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S.Pat. No. 4,820,508).

Useful dosages of the polypeptides can be determined by comparing theirin vitro activity, and in vivo activity in animal models. Methods forthe extrapolation of effective dosages in mice, and other animals, tohumans are known to the art; for example, see U.S. Pat. No. 4,938,949.

The amount of the polypeptides, or an active salt or derivative thereof,required for use in treatment will vary not only with the particularsalt selected but also with the route of administration, the nature ofthe condition being treated and the age and condition of the patient andwill be ultimately at the discretion of the attendant physician orclinician.

The polypeptides may be conveniently formulated in unit dosage form. Inone embodiment, the invention provides a composition comprising apolypeptide formulated in such a unit dosage form.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

The polypeptides of the invention can also be administered incombination with other therapeutic agents, for example, other agentsthat are useful for treating autoimmune diseases, classical Hodgkinlymphoma, atherosclerosis, cardiovascular disease, neuropathic pain, orcertain amenable types of leukemia and lymphoma, such as T cell largegranular lymphocyte leukemia. Accordingly, in one embodiment theinvention also provides a composition comprising a polypeptide describedherein, at least one other therapeutic agent, and a pharmaceuticallyacceptable diluent or carrier. The invention also provides a kitcomprising a polypeptide of the invention, at least one othertherapeutic agent, packaging material, and instructions foradministering the polypeptide and the other therapeutic agent or agentsto an animal to treat an autoimmune disease, classical Hodgkin lymphoma,atherosclerosis, cardiovascular disease, neuropathic pain, or certainamenable types of leukemia and lymphoma, such as T cell large granularlymphocyte leukemia.

Certain embodiments of the invention will now be illustrated by thefollowing non-limiting Examples.

Example 1

Little is known of the mechanism(s) regulating IRF5 nuclearlocalization. Two functional nuclear localization signals (NLS) wereidentified and characterized in the IRF5 protein that were not conservedor homologous with NLS in other IRFs. The IRF5 polypeptide contains twoNLSs, one residing in the amino-terminus (PRRVRLK) (SEQ ID NO: 1), andthe other in the carboxyl-terminus (PREKKLI) (SEQ ID NO:2) (Barnes etal. (2002) Mol Cell Biol 22, 5721-5740). While both NLSs could signaltranslocation to the nucleus, each had a distinct function. Theamino-terminal NLS, located in the DNA binding domain, contributed tonuclear localization and retention, while the carboxyl-terminal NLS wasresponsive to virus-induced nuclear translocation but not retention.These two NLSs are solely responsible for nuclear localization andtransactivation of target promoters as a mutant lacking both NLSs waslocalized in the cytoplasm and non-functional (Barnes et al. (2002) MolCell Biol 22, 5721-5740). Data from crystallography studies (Chen et al.(2008) Nat Struct Mol Biol 15, 1213-1220) and mutational analyses(Barnes et al. (2002) Mol Cell Biol 22, 5721-5740) indicate that theamino-terminal NLS is masked by an intramolecular interaction orassociation with another protein and post-translational modification isnecessary for exposure and retention of IRF5 in the nucleus, along withenhancement of its transactivating potential. IRF5 also contains afunctional nuclear export signal that controls shuttling between thecytoplasm and nucleus. As described herein, cell permeable peptideinhibitors directed at each of these NLS were designed in order tospecifically inhibit IRF5 nuclear translocation.

The amino (5′)- and carboxyl (3′)-terminal NLS sequences of IRF5, alongwith additional controls to confirm specific peptide function versusnon-specific cationic peptide function (Table 1), were synthesized andconjugated to a protein transduction (PTD) sequence in order to renderthe peptide cell permeable (Bowdish et al. (2004) J Immunol 172,3758-3765). NLS sequences are cationic peptides that accumulate withincells when added exogenously. Peptide sequences were synthesized usingLifeTein Peptide Synthesis Services. Peptides were solubilized in PBSand tested over a concentration range (0.025, 0.25, 2.5 and 25 μM) inhuman immortalized and/or primary cells.

TABLE 1 IRF5 NLS peptide inhibitors and controls. IRF5 5′NLSDRQIKIWFQNRRMKWKKPRRVRLK (SEQ ID NO: 3) IRF5 3′NLSDRQIKIWFQNRRMKWKKPREKKLI (SEQ ID NO: 4) PTDDRQIKIWFQNRRMKWKK (SEQ ID NO: 5) Scrambled 5′NLSDRQIKIWFQNRRMKWKKPKRRRLV (SEQ ID NO: 6) Scrambled 3′NLSDRQIKIWFQNRRMKWKKPIKRLKE (SEQ ID NO: 7)

FITC (Fluorescein isothiocyanate)-conjugated peptides were synthesizedto examine cell penetrance by fluorescent microscopy and flow cytometry.In general, cells were incubated with inhibitors for 4 hours beforeanalysis or stimulation. Inhibitor titrations, cytotoxicity, and cellcycle assays were performed to determine the optimal inhibitorconcentration that does not affect cell growth. The ability of eachpeptide to inhibit IRF5 nuclear localization was then determined on theAmnis Imagestream imaging flow cytometer. Example 4 also describessimilar experiments (see, FIGS. 1A-C and 2A-B).

Example 2

The transcription factor interferon regulatory factor 5 (IRF5) haspreviously been implicated in the onset of the autoimmune disordersystemic lupus erythrematosus (SLE). Elevated levels of inflammatorycytokines are a common characteristic of SLE, and are believed tocontribute to both autoantibody production and wide spread inflammation.Upon activation, cytoplasmic IRF5 translocates to the nucleus toinitiate pro-inflammatory gene transcription. To achieve nucleartranslocation, IRF5 relies on two nuclear localization signals locatedin the N′ and C′ termini of the protein. To investigate the therapeuticpotential of IRF5 inhibition, two unique cell-penetrating peptides havebeen developed (SEQ ID NO:3 and SEQ ID NO:4). Upon treatment with theseinhibitors, IRF5 is excluded from the nucleus, while IRF7 and NFκBnuclear translocation were unaffected following activation. The impactof IRF5 inhibition has also been investigated in a variety of cell linesas well as primary peripheral blood mononuclear cells. The inhibitorsshow no impact on cell cycle, viability, or IRF5 protein levels. As IRF5has been linked to expression of IgG subtypes in mice, the IRF5inhibitors were used to examine the impact of IRF5 inhibition in theRamos B cell line. Interestingly, no impact of the inhibitors on surfaceIgG expression was found. In THP1 cells, however, a marked reduction ininflammatory cytokine expression was seen following stimulation with LPSand IFNγ. These data highlight the usefulness of targeting IRF5 in orderto reduce the inflammatory signature characteristic of SLE patients.

Example 3

The transition of naïve B cells to effector B cells is dependent on alarge transcription factor network, which mediates both effector B celldifferentiation and function. The full repertoire of transcriptionfactors involved in this process is not known, yet dysregulation of thistranscription factor network can result in altered B cell function andautoimmunity. It appears that the transcription factor, interferonregulatory factor 5 (IRF5), is involved in the development of effector Bcells. Irf5^(−/−) mice have previously been reported to have reducedplasma B cells, as well as reduced serum IgG subtypes. It remainsunclear, however, what role IRF5 may play in human B cell developmentand function. Significant levels of IRF5 in B cells translocate to thenucleus following stimulation with anti-IgM antibody and CpG. In orderto characterize the role of IRF5 in human B cells, IRF5 ChIP-Seq havebeen performed in both primary naïve B cells and Ramos B cells eithermock or anti-IgM and CpG treated. Genes associated with plasma B celldevelopment were significantly enriched following activation, suggestingIRF5 plays a critical role in the differentiation of plasma B cells. Tofurther characterize the role of IRF5 in primary human B cells,siRNA-mediated knockdown of IRF5 has been performed. Knockdown of IRF5did not show significant impact on cell viability, however, reducedinflammatory cytokine expression, decreased plasmablast differentiation,and decreased IgG subtype production were seen. These data highlight themulti-functional role of IRF5 in regulating both human B celldifferentiation and function.

Example 4 Patients and Methods

Human Peripheral Blood Mononuclear Cell (PBMC) Isolation.

Approximately 200-350 milliliters (mLs) of blood was drawn fromconsented donors and subsequently diluted 1:1 with PBS without Calciumor Magnesium (PBS-Ca²⁺—Mg²⁺). Alternatively, prepared buffy coatsisolated from healthy donors were purchased from the New York BloodCenter and diluted 2-fold in PBS-Ca²⁺—Mg²⁺. Approximately 35 mLs ofdiluted blood was layered onto 15 mLs of Ficoll at room temperature in50 mL tubes. The blood Ficoll suspension was then centrifuged for 30minutes at 400 G with no brake at 21° C. in a swing bucket centrifuge.Following centrifugation, the serum layer was removed through pipetaspiration and the buffy coat layer transferred to a fresh 50 mL tube.The resulting buffy coat layer was washed two times in PBS-Ca²⁺-Mg²⁺ andresuspended in PBS-Ca²⁺—Mg²⁺ supplemented with 5% FBS. Isolated PBMCswere immediately utilized for B cell experiments. All experiments wereapproved by the Rutgers Biomedical and Health Sciences IRB and theFeinstein Institute for Medical Research IRB. Informed consent wasobtained from all healthy donors and experiments were performed inaccordance with Institution guidelines.

Peptide Inhibitor Titration and Uptake.

Ramos B cells or THP1 cells were treated with 0.025, 0.25, 2.5, and 25uM of FITC conjugated peptide inhibitors for 2 hours. Cells were washed,stained with propidium iodide, and immediately analyzed by flowcytometry for uptake of FITC peptide. For uptake experiments in PBMCs,cells were incubated with 10 uM of either mock, scrambled, N-term′ NLS,or C-term′ NLS FITC conjugated inhibitor for 2 hours. Cells were thenwashed and blocked as previously outlined, and subsequently stained withCD19-BV510 and CD14-PE (Biolegend Catalog#301806). Cells were fixed,stained with DRAQ5 nuclear dye and analyzed by imagestream for uptake ofFITC peptide.

Quantitative Real-Time PCR.

RNA was prepared from nucleofected primary B cells by Trizol®(guanidinium thiocyanate-phenol-chloroform extraction method) isolation.Following RNA isolation, cDNA was prepared followed by quantitativereal-time PCR (qPCR) using specific primer sets. Each sample was assayedin replicates of three, per primer set used. The threshold values(C_(T)) were averaged over each sample replicated, followed bynormalization via the ΔΔC_(T) method to a housekeeping gene such asBeta-actin.

Imaging Flow Cytometry.

PBMCs were isolated as previously outlined, and treated with inhibitorfor 1-2 hours, followed by stimulation with R848 (500 ng/mL) or SLESerum (2%). PBMCs were then stained for CD19 (BD Biosciences Catalog#:562847) or CD14 (Biolegend Catalog #: 301806) for 1 hour, after which,cells were fixed overnight in 1% paraformaldehyde. The following daycells were and permeabilized in 0.1% Triton-X-100. Permeabilized cellswere blocked in 5% BSA solution and subsequently stained for IRF5 (AbcamCatalog #: ab193245). Cells were washed and fixed in 1%paraformaldehyde. Prior to acquisition, the nuclear dye DRAQ5 was addedat a 1:50 dilution. Images were acquired on the Amnis Image Stream usingthe 40× objective. Nuclear translocation was quantified in the AmnisIDEAS software suite. Cells were first filtered through the brightfieldarea vs brightfield aspect ratio gate to exclude non-viable and doubletevents. Following which a similar gate of the DRAQ5 nuclear channel wasused. This added an extra measure of stringency for cell viability.Images were gated on either CD14⁺IRF5⁺ or CD19⁺IRF5⁺ events, followed bygating on images with a Gradient RMS of greater than 20 on the DRAQ5channel. This was done to select images with a high level of clarity.Finally, IRF5 nuclear translocation was determined through use of thesimilarity score feature contrasting IRF5 staining with DRAQ5 staining.A similarity score of greater than or equal to 2 was considered atranslocation event.

Cell Fractionation.

Cells were fractionated according to manufacturer's protocol (CellSignaling, Cell fractionation kit, #9038). Following fractionation,lysates were sonicated and boiled. Nuclear fraction was analyzed byWestern blot as follows: 30 μL of lysate was loaded onto a 3-8% NuPAGE®Novex® Tris-Acetate gel (Life Technologies, #EA0378BOX), and transferredonto a 0.45 μm nitrocellulose membrane (Bio-Rad Laboratories). Membranewas blocked in TBS/0.25% Tween 20 containing 5% BSA for 1 h at RT andincubated overnight at 4° C. with α-IRF5 antibody (Cell Signaling,#13496) followed by HRP-conjugated secondary antibody (Cell Signaling,α-rabbit #7074S). The nuclear fraction was confirmed using Lamin B1(Cell Signaling, #15068). Membrane was incubated with Clarity™ ECLWestern Blotting Substrate (Bio-Rad Laboratories) and chemiluminescencedetected with a ChemiDoc™ MP Imaging System (Bio-Rad Laboratories). ThePageRuler™ Plus Prestained Protein Ladder (ThermoFisher Scientific) wasused for size reference.

Statistical Analysis.

For experiments shown, >=3 experimental replicates were used unlessotherwise noted. Student's t-test was used for comparisons between twosamples with normal distribution. Prior to test, graph kurtosis wasanalyzed to ensure normal distribution. For comparisons of one factorover multiple groups, One-Way ANOVA was performed with Tukey's post-hoctest for significance. For comparisons of multiple factors over multiplegroups, Two-Way ANOVA was performed with Tukey's post-hoc test forsignificance.

Results

Design of IRF5 Peptide Inhibitors.

Given the association of IRF5 with the onset of several autoimmunedisorders, as well as work that has been done in establishing the roleof IRF5 in B cell proliferation and antibody production, it wasinvestigated whether therapeutic inhibition of IRF5 nucleartranslocation was feasible. To accomplish this, peptide inhibitors ofthe IRF5 amino (N′)-terminal and carboxyl (C′)-terminal nucleartranslocation signals (NLS) were designed. Precedence for this strategycomes from previous NFκB inhibitors, which have been proven to beeffective in inhibiting NFκB-mediated transcriptional activity (Lin, Y.Z., et al., J Biol Chem, 1995. 270(24): p. 14255-8; Mallavia, B., etal., Am J Pathol, 2013. 182(5): p. 1910-21; Orange, J. S. and M. J. May,Cell Mol Life Sci, 2008. 65(22): p. 3564-91; Zhang, L., et al., ProcNatl Acad Sci USA, 1998. 95(16): p. 9184-9). To transduce the cellmembrane, IRF5 NLS sequences were combined with a protein transductiondomain (PTD). The PTD has been previously shown to facilitate cellpermeability of small peptides (see, Orange, J. S. and M. J. May, CellMol Life Sci, 2008. 65(22): p. 3564-91). Design of the N′-terminal NLSinhibitor was accomplished by merging the PTD sequence“DRQIKIWFQNRRMKWKK (SEQ ID NO:5)” with the IRF5 NLS sequence of “PRRVRLK(SEQ ID NO: 1)”. The N′-terminal NLS sequence used corresponded to aminoacids 12 thru 18 of IRF5 variant 5. A similar strategy was used for theC′-terminal NLS inhibitor, whereby the PTD sequence was merged with the“PREKKLI (SEQ ID NO:2)” C′-terminal NLS sequence, corresponding to aminoacids 408 thru 441 of IRF5 variant 5. A control peptide of the PTDsequence alone and one merged to a scrambled NLS sequence of “PKRRRLV(SEQ ID NO:8)” were also designed (see, Table 1).

IRF5 Peptide Inhibitors Readily Enter the Cell and have Low AssociatedToxicity.

IRF5 peptide inhibitors were conjugated to FITC moieties to measurecellular uptake over a dose-dependent response. Ramos B cells weretreated with increasing concentrations of FITC-conjugated inhibitor for2 hours. Cells were washed and subsequently treated with propidiumiodide to quantify cell death. While at the lower doses of 0.025 and0.25 uM minimal uptake was seen, at 2.5 uM however, greater than 95% ofcells were positive for the FITC-conjugated inhibitor (FIG. 1A). Inaddition, less than 3% of cells stained positive for propidium iodide,indicating low toxicity for the inhibitor at this dose. At the 25 uMdose, greater than 90% of cells had taken up the inhibitor, while bothscrambled and N′-terminal inhibitors showed minimal associated toxicity.However, no significant increase in uptake was seen between 2.5 uM and25 uM concentrations of inhibitor. To ensure no impact on cell viabilityfrom treatment with the inhibitors, Ramos B cells were treated withvarying concentrations of inhibitor as used previously for the titrationexperiments for 24 hours, and tryphan blue staining was subsequentlyperformed. Similar to our previous titration results, minimal toxicityassociated with the inhibitors was observed at concentrations up to 2.5uM. However, at 25 uM a decrease in viability was noted for thescrambled inhibitor (FIG. 1B). Additionally, to ensure the peptideinhibitors did not have significant off target effects, measured cellcycle progression was measured in Ramos B cells following treatment withincreasing concentrations of inhibitor. Cell cycle was measured throughpropidium iodide incorporation, and quantified through flow cytometry.No significant difference in cell cycle progression was seen at any ofthe dosages of the peptide inhibitor (FIG. 1C). Next, the uptakeexperiments in primary peripheral blood mononuclear cells isolated fromhealthy donors was sought to be confirmed. Therefore, imaging flowcytometry was utilized to confirm uptake of the inhibitors in primaryPBMCs, followed by gating on B cells and monocytes. Isolated PBMCs weretreated with 2.5 uM of either mock, N′-terminal, or Scrambled NLSinhibitors for an hour, and subsequently stained with CD19 to demarcateB cells and CD14 to identify monocytes. Both B cells and monocytes hadindeed successfully taken up the peptide inhibitor within 1 hour (FIG.1D). Interestingly, monocytes showed a much higher level of uptake thanthat seen in B cells. To determine if it would be possible to increaseuptake of the inhibitor in B cells, uptake following incubation with 10uM for 1 hour was measured. B cells showed a slight increase in uptakefollowing treatment with 10 uM of inhibitor, while monocytes continuedto have higher levels of uptake than B cells. This suggested it may bepossible to achieve cell type-specific activity of the inhibitor throughvaried dosage.

IRF5 Peptide Inhibitor Blocks IRF5 Nuclear Translocation in Both B Cellsand Monocytes.

IRF5 nuclear translocation has been shown to be consequential toincreased IFN secretion and antibody production, therefore, inhibitionof nuclear translocation would have significant therapeutic value. AsIRF5 has been implicated in the onset of autoimmune diseases in bothmonocytes and B cells, it was sought to be determined if IRF5 nucleartranslocation could be inhibited in both cell populations followingstimulation with the TLR7 agonist R848. Due to the use of PBMCs, R848was utilized for stimulation as it is recognized by TLR7 on bothmonocytes and B cells. Isolated PBMCs were cultured in the presence ofeither mock, scrambled, N′-terminal, or C′-terminal NLS inhibitor for 1hour followed by stimulation with 500 ng/mL of R848 for 2 hours. Asignificant reduction in IRF5 nuclear translocation was seen in bothmonocytes and B cells following treatment with the N′-terminal NLSinhibitor in comparison to the scrambled control (FIGS. 2A,B).Representative images are shown in FIG. 2C. In monocytes, roughly a3-fold reduction, and an approximate 2-fold reduction in B cells wasseen between scrambled control and the N′-terminal inhibitor. Incontrast, the-C′-terminal inhibitor showed only slightly reduced nucleartranslocation in monocytes that failed to achieve significance, while inB cells, the C′-terminal inhibitor showed no inhibition of IRF5 nucleartranslocation. To further confirm a block in nuclear translocation ofIRF5, cell fractionation was performed on isolated primary monocyteswhich were treated with inhibitor and subsequently stimulated with R848.Reduced levels of IRF5 were seen in the nucleus in the presence ofeither the N′- or C′-terminal inhibitors (FIGS. 2D,E).

IRF5 Peptide Inhibitors are Specific to IRF5.

As inhibition of IRF5 activity achieved through the peptide inhibitorsmay rely on saturation of nuclear translocation machinery, it remainedpossible other transcription factors would be similarly impacted.Specifically, transcription factors which require nuclear translocationand have overlapping transcriptional targets with IRF5, such as NFκB,seemed to be the most logical candidates to assay. Therefore, weexamined NFκB nuclear translocation following treatment with the IRF5peptide inhibitors and subsequent R848 stimulation in both B cells andmonocytes. No significant impact was seen on NFκB nuclear translocationin either B cells or monocytes in the presence of the inhibitor (FIG.3A). Overall nine IRF transcription factors exist in humans, with allnine having high similarity. To determine if other members of the IRFfamily of transcription factors were impacted by the IRF5 peptideinhibitors, nuclear translocation of another IRF transcription factorimportant in inflammatory cytokine expression was measured. IRF7 isknown to be important in regulating interferon secretion in plasmacytoiddendritic cells (pDCs), with overlapping functions with IRF5. Therefore,IRF7 nuclear translocation was quantified in pDCs following treatmentwith the inhibitors and also stimulation with R848. No significantimpact was seen on IRF7 nuclear translocation (FIG. 3B).

IRF5 Peptide Inhibitors Reduce Expression of Inflammatory CytokineExpression.

Immediately following nuclear translocation, IRF5 upregulatespro-inflammatory cytokine expression in both monocytes and B cells. InSLE it is believed that increased levels of inflammatory cytokinescontribute to altered B cell differentiation, as well as increasedsystemic inflammation (Banchereau, J. and V. Pascual, Immunity, 2006.25(3): p. 383-92; Tackey, et al., Lupus, 2004. 13(5): p. 339-43). It hasbeen previously shown that both monocytes and B cells release elevatedlevels of inflammatory cytokines such as IL6 (Stone, R. C., et al.,Arthritis Rheum, 2012. 64(3): p. 788-98; Tackey, et al., Lupus, 2004.13(5): p. 339-43). Therefore, blockade of IRF5 nuclear translocationshould ultimately impact inflammatory cytokine production. IL6 and IL10production were initially examined in the Ramos B cell and THP monocytecell lines, following stimulation. Both Ramos and THP1 cell lines werepre-treated with inhibitor for 1 hour, followed by LPS stimulation ofTHP1 cells and anti-IgM plus CpG-B stimulation of Ramos B cells for 2hours. For both cell types, IL6 and IL10 production were mostsignificantly reduced following treatment with the N′-terminal inhibitor(data not shown). In the case of monocytes, a 2-fold inhibition of IL6expression was seen, whereas in Ramos B cells, a 3-fold reduction inexpression was noted. IL6, IFNa, and IL10 production were examined nextin isolated PBMCs treated with inhibitor for 1 hour, followed by R848stimulation for 2 hours. All three cytokines were found to besignificantly reduced following treatment with the N′-terminal inhibitor(FIG. 4). A 70% reduction in IL6, a 35% reduction in IL10, and a 52%reduction in IFNa expression was seen after treatment with theN′-terminal inhibitor. This demonstrates inhibition of IRF5 nucleartranslocation results in a direct reduction in inflammatory cytokineexpression.

IRF5 Peptide Inhibitors are Effective in Reducing IRF5 NuclearTranslocation in PBMCs from SLE Patients.

Serum from SLE patients results in increased IRF5 nuclear translocationfollowing addition to healthy PBMCs. Serum stimulation of healthy PBMCsmost likely triggers multiple activation pathways, and would represent amore disease relevant stimuli than those of purified TLR ligands. Todetermine if the peptide inhibitors described herein would be effectivefollowing addition of complex stimuli such as SLE serum, isolated PBMCsfrom healthy donors were treated with the peptide inhibitors followed bystimulation with SLE serum from individual patients. IRF5 nucleartranslocation was again assayed through imaging flow cytometry,following which a significant reduction in IRF5 nuclear translocationwas observed (FIG. 5). An inhibitor utilized for the treatment of SLEshould also demonstrate an ability to block basal IRF5 nucleartranslocation in SLE patients. SLE patients have been shown to have highlevels of basal IRF5 nuclear translocation. To determine if thesepeptide inhibitors will be effective in reversing basal elevated IRF5nuclear translocation, PBMCs will be isolated from SLE patients havingSLEDAI scores=0, >0<4, >4, and with active flares. Data thus far supportthat pre-treatment with the inhibitors results in a significant decreasein basal IRF5 nuclear translocation in both SLE monocytes and B cells.

Example 5

In vivo murine experiments were performed using NZB/W F1 mice, whichmodel spontaneous lupus, and the IRF5 peptide inhibitor(DRQIKIWFQNRRMKWKKPRRVRLK (SEQ ID NO:3)) (Life Tein, LLC). Specifically,treatment groups were examined at 8 weeks of age and at 27 weeks of age,as described in Table 2 below.

TABLE 2 Treatment Groups Control (Ctrl) no treatment (n = 6) 8 Weeks ofAge 100 μg 100 μg/mouse/injection on Day 0, Day 1, Day 4, Day 7 and Day14 (n = 6) 200 μg 200 μg/mouse/injection on Day 0, Day 1, Day 4, Day 7and Day 14 (n = 6) (Data not presented) Multiple 100 μg/mouse/injectionthree times per week for 5 weeks (n = 6). (Data not presented; Dosingwas discontinued after blood spotted in urine at week 12) 27 Weeks ofAge 100 μg 100 μg/mouse/injection on Day 0, Day 1, Day 4, Day 7 and Day14 (n = 6)  50 μg 50 μg/mouse/injection on Day 0, Day 1, Day 4, Day 7and Day 14 (n = 6) (Data not presented) Multiple 30 μg/mouse/injectionthree times per week for 3 weeks (n = 6). (Data not presented)

The results of these experiments are shown in FIGS. 6-10.

Example 6

The specific binding of the 5′-NLS inhibitor (DRQIKIWFQNRRMKWKKPRRVRLK(SEQ ID NO:3)) and the 3′-NLS inhibitor (DRQIKIWFQNRRMKWKKPREKKLI (SEQID NO:4)) (Life Tein, LLC) to human recombinant full-length IRF5 wasinvestigated. Specifically, g/mL recombinant IRF5 was immobilized on theBiacore Sensor Chip CM5 at a flow rate of L/min using manual injection(450RU). Running buffer was filtered 1×PBS with 0.05% P20 run at a flowrate of 30 μl/min. Contact time—60 sec; Dissociation time—120 sec.Samples were run on a Biacore-T200. Specific binding of the 5′-NLSinhibitor to human recombinant full-length IRF5 is shown in FIG. 11.

All documents cited herein are incorporated by reference. While certainembodiments of invention are described, and many details have been setforth for purposes of illustration, certain of the details can be variedwithout departing from the basic principles of the invention.

The use of the terms “a” and “an” and “the” and similar terms in thecontext of describing embodiments of invention are to be construed tocover both the singular and the plural, unless otherwise indicatedherein or clearly contradicted by context. The terms “comprising,”“having,” “including,” and “containing” are to be construed asopen-ended terms (i.e., meaning “including, but not limited to”) unlessotherwise noted. Recitation of ranges of values herein are merelyintended to serve as a shorthand method of referring individually toeach separate value falling within the range, unless otherwise indicatedherein, and each separate value is incorporated into the specificationas if it were individually recited herein. In addition to the orderdetailed herein, the methods described herein can be performed in anysuitable order unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein, is intended merely to betterilluminate embodiments of invention and does not necessarily impose alimitation on the scope of the invention unless otherwise specificallyrecited in the claims. No language in the specification should beconstrued as indicating that any non-claimed element is essential to thepractice of the invention.

What is claimed is:
 1. A polypeptide comprising a cell penetratingpeptide sequence and an interferon regulatory factor 5 (IRF5) nuclearlocalization signal (NLS) sequence, wherein the cell penetrating peptidesequence facilitates cellular uptake of the polypeptide.
 2. Thepolypeptide of claim 1, wherein the polypeptide comprises formula (I):X-Y-Z  (I) wherein X is a cell penetrating peptide sequence; Y is alinking group; and Z is an IRF5 NLS sequence.
 3. (canceled)
 4. Thepolypeptide of claim 1, wherein the cell penetrating peptide sequencecomprises a sequence at least 90% identical to DRQIKIWFQNRRMKWKK (SEQ IDNO: 5), AAVALLPAVLLALLAP (SEQ ID NO: 9), GRKKRRQRRRPPQ (SEQ ID NO: 10),CSIPPEVKFNKPFVYLI (SEQ ID NO: 11), KKWKMRRNQFWVKVQRG (SEQ ID NO: 12),KLLKLLLKLWLKLLKLLL (SEQ ID NO: 13), _INLKALAALAKKIL (SEQ ID NO: 14),RQIKIWFQNRRMKWKKGG (SEQ ID NO: 15) or GWTLNSAGYLLGKINLKALAALAKKIL (SEQID NO: 16).


5. The polypeptide of claim 4, wherein the cell penetrating peptidesequence is selected from the group consisting of DRQIKIWFQNRRMKWKK (SEQID NO:5), AAVALLPAVLLALLAP (SEQ ID NO:9), GRKKRRQRRRPPQ (SEQ ID NO: 10),CSIPPEVKFNKPFVYLI (SEQ ID NO: 11), KKWKMRRNQFWVKVQRG (SEQ ID NO: 12),KLLKLLLKLWLKLLKLLL (SEQ ID NO: 13), INLKALAALAKKIL (SEQ ID NO: 14),RQIKIWFQNRRMKWKKGG (SEQ ID NO:15) and GWTLNSAGYLLGKINLKALAALAKKIL (SEQID NO: 16).
 6. (canceled)
 7. The polypeptide of claim 1, wherein theIRF5 NLS sequence comprises a sequence at least 90% identical to PRRVRLK(SEQ ID NO: 1) or PREKKLI (SEQ ID NO:2).
 8. The polypeptide of claim 7,wherein the IRF5 NLS sequence is PRRVRLK (SEQ ID NO: 1) or PREKKLI (SEQID NO:2). 9-10. (canceled)
 11. The polypeptide of claim 1, whichcomprises a sequence at least 90% identical to: DRQIKIWFQNRRMKWKKPRRVRLK(SEQ ID NO: 3) or DRQIKIWFQNRRMKWKKPREKKLI (SEQ ID NO: 4).


12. (canceled)
 13. The polypeptide of claim 11, which comprises asequence at least 99% identical to SEQ ID NO:3 or SEQ ID NO:4.
 14. Thepolypeptide of claim 13, which comprises SEQ ID NO:3 or SEQ ID NO:4.15-19. (canceled)
 20. The polypeptide of claim 1, which polypeptide isan inhibitor of interferon regulatory factor 5 (IRF5).
 21. Thepolypeptide of claim 1, which polypeptide is an inhibitor of interferonregulatory factor 5 (IRF5) nuclear localization. 22-25. (canceled) 26.The polypeptide of claim 1, wherein the polypeptide comprises a chemicalmodification.
 27. A nucleic acid sequence encoding the polypeptide ofclaim
 1. 28. A pharmaceutical composition that comprises a polypeptideof claim 1 and a pharmaceutically acceptable carrier.
 29. A method ofinhibiting interferon regulatory factor 5 (IRF5) in a patient in needthereof, comprising administering to the patient a therapeuticallyeffective amount of a polypeptide of claim
 1. 30. (canceled)
 31. Amethod for treating an autoimmune disease, classical Hodgkin lymphoma,atherosclerosis, cardiovascular disease, neuropathic pain, leukemia orlymphoma in a patient in need of such treatment, comprisingadministering to the patient a therapeutically effective amount of apolypeptide of claim
 1. 32. The method of claim 31, wherein the methodis for treating an autoimmune disease, and wherein the autoimmunedisease is systemic lupus erythematosus (SLE), systemic sclerosis(scleroderma), polymyositis/dermatomyositis, Crohn's disease, rheumatoidarthritis, periodontitis, SLE-associated atherosclerosis, Sjögren'ssyndrome, autoimmune encephalomyelitis, sarcoidosis, Behçet's disease,myasthenia gravis, lupus nephritis, inflammatory bowel disease,ankylosing spondylitis, primary biliary cirrhosis, colitis, juvenileidiopathic arthritis, pulmonary fibrosis, antiphospholipid syndrome, orpsoriasis.
 33. The method of claim 32, wherein the autoimmune disease issystemic lupus erythematosus (SLE).
 34. (canceled)
 35. The method ofclaim 31, further comprising administering a second therapeutic agent.36-47. (canceled)
 48. A kit comprising a polypeptide as described inclaim 1, at least one other therapeutic agent, and instructions foradministering the polypeptide and the other therapeutic agent(s) to ananimal to treat an autoimmune disease, classical Hodgkin lymphoma,atherosclerosis, cardiovascular disease, neuropathic pain, leukemia orlymphoma.